estimation-nephron-number-whole-kidney-using-acid-maceration

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

The University of Mississippi Medical Center

Nuclear hormone receptors of the NR4A subgroup have been implicated in cancer, atherosclerosis, and metabolic disease. However, little is known about the role of these receptors in kidney health or disease. Nr4a1-deficient rats (Nr4a1(-/-)) developed on a genetic background susceptible to kidney injury (fawn-hooded hypertensive rat [FHH]) were evaluated for BP, proteinuria, renal function, and metabolic parameters from 4 to 24 weeks-of-age. By week 24, Nr4a1(-/-) rats exhibited significantly higher proteinuria (approximately 4-fold) and decreased GFR compared with FHH controls. The severity of tubular atrophy, tubular casts, and interstitial fibrosis increased significantly in Nr4a1(-/-) rats and was accompanied by a large increase in immune cell infiltration, predominantly macrophages and to a lesser extent T cells and B cells. Global transcriptome and network analyses at weeks 8, 16, and 24 identified several proinflammatory genes and pathways differentially regulated between strains. Bone marrow crosstransplantation studies demonstrated that kidney injury in Nr4a1(-/-) rats was almost completely rescued by bone marrow transplanted from FHH controls. In vitro, macrophages isolated from Nr4a1(-/-) rats demonstrated increased immune activation compared with FHH-derived macrophages. In summary, the loss of Nr4a1 in immune cells appears to cause the increased kidney injury and reduced renal function observed in the Nr4a1(-/-) model. 

Genetic susceptibility and loss of Nr4a1 enhances macrophage-mediated renal injury in CKD.

Some studies have reported up to 40% of patients born with a single kidney develop hypertension, proteinuria, and in some cases renal failure. The increased susceptibility to renal injury may be due, in part, to reduced nephron numbers. Notably, children who undergo nephrectomy or adults who serve as kidney donors exhibit little difference in renal function compared with persons who have two kidneys. However, the difference in risk between being born with a single kidney versus being born with two kidneys and then undergoing nephrectomy are unclear. Animal models used previously to investigate this question are not ideal because they require invasive methods to model congenital solitary kidney. In this study, we describe a new genetic animal model, the heterogeneous stock-derived model of unilateral renal agenesis (HSRA) rat, which demonstrates 50%-75% spontaneous incidence of a single kidney. The HSRA model is characterized by reduced nephron number (more than would be expected by loss of one kidney), early kidney/glomerular hypertrophy, and progressive renal injury, which culminates in reduced renal function. Long-term studies of temporal relationships among BP, renal hemodynamics, and renal function demonstrate that spontaneous single-kidney HSRA rats are more likely than uninephrectomized normal littermates to exhibit renal impairment because of the combination of reduced nephron numbers and prolonged exposure to renal compensatory mechanisms (i.e., hyperfiltration). Future studies with this novel animal model may provide additional insight into the genetic contributions to kidney development and agenesis and the factors influencing susceptibility to renal injury in individuals with congenital solitary kidney. 

Nephron Deficiency and Predisposition to Renal Injury in a Novel One-Kidney Genetic Model.

Ischemia-reperfusion (IR) injury is the most common cause of AKI. The susceptibility to develop AKI varies widely among patients. However, little is known about the genes involved. 20-Hydroxyeicosatetraenoic acid (20-HETE) has an important role in the regulation of renal tubular and vascular function and has been implicated in IR injury. In this study, we examined whether a deficiency in the renal formation of 20-HETE enhances the susceptibility of Dahl salt-sensitive (SS) rats to ischemic AKI. Transfer of chromosome 5 containing the CYP4A genes responsible for the formation of 20-HETE from the Brown Norway (BN) rat onto the SS genetic background increased renal 20-HETE levels after ischemia and reduced plasma creatinine levels (±SEM) 24 hours after IR from 3.7±0.1 to 2.0±0.2 mg/dl in an SS.5(BN)-consomic strain. Transfer of this chromosome also prevented the secondary decline in medullary blood flow and ischemia that develops 2 hours after IR in the susceptible SS strain. Blockade of the synthesis of 20-HETE with HET0016 reversed the renoprotective effects in SS.5(BN) rats. Similar results were observed in an SS.5(Lew)-congenic strain, in which a smaller region of chromosome 5 containing the CYP4A genes from a Lewis rat was introgressed onto the SS genetic background. These results indicate that 20-HETE has a protective role in renal IR injury by maintaining medullary blood flow and that a genetic deficiency in the formation of 20-HETE increases the susceptibility of SS rats to ischemic AKI. 

Deficiency in the Formation of 20-Hydroxyeicosatetraenoic Acid Enhances Renal Ischemia-Reperfusion Injury.

There is little clinical data of how hypertension may influence individuals with nephron deficiency in the context of being born with a single kidney. We recently developed a new rat model (the heterogeneous stock-derived model of unilateral renal agenesis rat) that is born with a single kidney and exhibits progressive kidney injury and decline in kidney function with age. We hypothesized that DOCA-salt would induce a greater increase in blood pressure and therefore accelerate the progression of kidney injury in rats born with a solitary kidney compared with rats that have undergone unilateral nephrectomy. Time course evaluation of blood pressure, kidney injury, and renal hemodynamics was performed in the following six groups of animals from weeks 13 to 18: 1) DOCA-treated rats with a solitary kidney (DOCA+S group), 2) placebo-treated rats with a solitary kidney, 3) DOCA-treated control rats with two kidneys (DOCA+C group), 4) placebo-treated control rats with two kidneys, 5) DOCA-treated rats with two kidneys that underwent uninephrectomy (DOCA+UNX8 group), and 6) placebo-treated rats with two kidneys that underwent uninephrectomy. DOCA+S rats demonstrated a significant rise (P < 0.05) in blood pressure (192 ± 4 mmHg), proteinuria (205 ± 31 mg/24 h), and a decline in glomerular filtration rate (600 ± 42 μl·min(-1)·g kidney weight(-1)) relative to the DOCA+UNX8 (173 ± 3 mmHg, 76 ± 26 mg/24 h, and 963 ± 36 μl·min(-1)·g kidney weight(-1)) and DOCA+C (154 ± 2 mmHg, 7 ± 1 mg/24 h, and 1,484 ± 121 μl·min(-1)·g kidney weight(-1)) groups. Placebo-treated groups showed no significant change among the three groups. An assessment of renal injury markers via real-time PCR/Western blot analysis and histological analysis was concordant with the measured physiological parameters. In summary, congenital solitary kidney rats are highly susceptible to the induction of hypertension compared with uninephrectomized rats, suggesting that low nephron endowment is an important driver of elevated blood pressure, hastening nephron injury through the transmission of elevated systemic blood pressure and thereby accelerating decline in kidney function.

Spontaneous one-kidney rats are more susceptible to develop hypertension by DOCA-NaCl and subsequent kidney injury compared with uninephrectomized rats.

The kidneys play a vital role in the excretion of waste products and the regulation of electrolytes, maintenance of acid-base balance, regulation of blood pressure, and production of several hormones. Any alteration in the structure of the nephron (basic functional unit of the kidney) can have a major impact on the kidney's ability to work efficiently. Progressive decline in kidney function can lead to serious illness and ultimately death if not treated by dialysis or transplantation. While there have been numerous studies that implicate lower nephron numbers as being an important factor in influencing susceptibility to developing hypertension and chronic kidney disease, a direct association has been difficult to establish because of three main limitations: ) the large variation in nephron number observed in the human population; ) no established reliable noninvasive methods to determine nephron complement; and ) to date, nephron measurements have been done after death, which doesn't adequately account for potential loss of nephrons with age or disease. In this review, we will provide an overview of kidney structure/function, discuss the current literature for both humans and other species linking nephron deficiency and cardio-renal complications, as well as describe the major molecular signaling factors involved in nephrogenesis that modulate variation in nephron number. As more detailed knowledge about the molecular determinants of nephron development and the role of nephron endowment in the cardio-renal system is obtained, it will hopefully provide clinicians the ability to accurately identify people at risk to develop CKD/hypertension and lead to a shift in patient care from disease treatment to prevention.

Nephron number, hypertension, and CKD: physiological and genetic insight from humans and animal models.

West Nile virus (WNV) infection results in a spectrum of neurological symptoms, ranging from a benign fever to severe WNV neuroinvasive disease with high mortality. Many who recover from WNV neuroinvasive infection present with long-term deficits, including weakness, fatigue, and cognitive problems. While neurons are a main target of WNV, other cell types, especially astrocytes, play an important role in promoting WNV-mediated central nervous system (CNS) damage. Conversely, it has been shown that cultured primary astrocytes secrete high levels of interferons (IFNs) immediately after WNV exposure to protect neighboring astrocytes, as well as neurons. However, how intrinsic responses to WNV in specific cell types and different regions of the brain modify immune protection is not fully understood. Here, we used a mouse ex vivo spinal cord slice culture (SCSC) and cerebellar slice culture (CSC) models to determine the innate immune responses specific to the CNS during WNV infection. Slices were prepared from the spinal cord and cerebellar tissue of 7⁻9-day-old mouse pups. Four-day-old SCSC or CSC were infected with 1 × 10³ or 1 × 10⁵ PFU of WNV, respectively. After 12 h exposure to WNV and 3 days post-infection in normal growth media, the pooled slice cultures were processed for total RNA extraction and for gene expression patterns using mouse Affymetrix arrays. The expression patterns of a number of genes were significantly altered between the mock- and WNV-treated groups, both in the CSCs and SCSCs. However, distinct differences were observed when CSC data were compared with SCSC. CSCs showed robust induction of interferons (IFNs), IFN-stimulated genes (ISGs), and regulatory factors. Some of the antiviral genes related to IFN were upregulated more than 25-fold in CSCs as compared to mock or SCSC. Though SCSCs had twice the number of dysregulated genes, as compared CSCs, they exhibited a much subdued IFN response. In addition, SCSCs showed astrogliosis and upregulation of astrocytic marker genes. In sum, our results suggest that early anti-inflammatory response to WNV infection in CSCs may be due to large population of distinct astrocytic cell types, and lack of those specialized astrocytes in SCSC may make spinal cord cells more susceptible to WNV damage. Further, the understanding of early intrinsic immune response events in WNV-infected ex vivo culture models could help develop potential therapies against WNV.

Differential Expression of Genes Related to Innate Immune Responses in Ex Vivo Spinal Cord and Cerebellar Slice Cultures Infected with West Nile Virus.

Article	Year
Genetic susceptibility and loss of Nr4a1 enhances macrophage-mediated renal injury in CKD. Journal of the American Society of Nephrology : JASN\| PubMed ID: 24722447	2014
Nephron Deficiency and Predisposition to Renal Injury in a Novel One-Kidney Genetic Model. Journal of the American Society of Nephrology : JASN\| PubMed ID: 25349207	2015
Deficiency in the Formation of 20-Hydroxyeicosatetraenoic Acid Enhances Renal Ischemia-Reperfusion Injury. Journal of the American Society of Nephrology : JASN\| PubMed ID: 25644108	2015
Spontaneous one-kidney rats are more susceptible to develop hypertension by DOCA-NaCl and subsequent kidney injury compared with uninephrectomized rats. American journal of physiology. Renal physiology\| PubMed ID: 26936874	2016
Nephron number, hypertension, and CKD: physiological and genetic insight from humans and animal models. Physiological genomics\| PubMed ID: 28130427	2017
Differential Expression of Genes Related to Innate Immune Responses in Ex Vivo Spinal Cord and Cerebellar Slice Cultures Infected with West Nile Virus. Brain sciences\| PubMed ID: 30586874	2018

Michael R. Garrett

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